The present invention relates to a method for regulating an MEKK/JNKK-contingent signal transduction pathway in a hematopoietic cell in order to regulate cytokine production by such cell.
Aggregation of the high-affinity Fc receptors for immunoglobulin E (IgE) (Fcxcex5RI) on the surface of mast cells initiates intracellular signal transduction pathways, involving the tyrosine phosphorylation of cellular proteins, activation of phospholipase Cxcex3, hydrolysis of phosphoinositide, increase in intracellular calcium, activation of protein kinase C and the stimulation of phosphatidylinositol 3-kinase. These signal transduction pathways are believed to be involved in the exocytic release of inflammatory mediators such as vasoactive amines, cytokines, and lipid metabolites. The production of cytokines by mast cells is a critical event that influences the pathogenesis of allergic inflammation in asthma and other allergic disorders.
In addition to the activation of phospholipase Cxcex3 and protein kinase C, which appears to be essential for the Fcxcex5RI-mediated release of preformed mediators, the aggregation of Fcxcex5RI on rat basophilic leukemia 2H3 (RBL-2H3) cells has been shown to induce histamine and leukotriene release. Except for the activation of the extracellular signal-regulated kinases/mitogen activated protein kinases (ERKs/MAPKs), however, the downstream consequences of early activation events in a signal transduction pathway leading to cytokine production are not well defined.
The extracellular signal-regulated kinases (ERKs), ERK1 and ERK2, are serine/threonine protein kinases that are activated through concomitant phosphorylation of tyrosine and threonine residues. Prior to the current invention, it was thought that ERKs were one of the intermediates in the signal transduction pathway leading to increases in gene transcription and proliferation, including cytokine gene transcription. ERKs phosphorylate specific transcription factors including members of the Ets family, such as Elk-1, and it has been reported that ERKs are activated via Fcxcex5RI on mast cells.
Despite the current understanding of early signal transduction events in hematopoietic cells, there remains a need to elucidate signal transduction pathways that specifically regulate cytokine production in such cells and to determine what molecules and/or functional elements of such molecules are responsible for regulating such cellular pathways. There is also a need for products and processes that permit the effective regulation of specific steps in such a signal transduction pathway. Regulation of specific steps of a signal transduction pathway which regulate cytokine production permits the implementation of predictable controls of such signal transduction in cells, thereby allowing modulation of the effects of cytokine production in diseases wherein such modulation can ameliorate disease pathogenesis.
The present invention generally relates to a method to regulate a novel signal transduction pathway to modulate the production of cytokines by a hematopoietic cell. The present inventors have identified an MEKK/JNKK-contingent signal transduction pathway which regulates the production of cytokines by a hematopoietic cell. Prior to the present invention, it was thought that signal transduction through the ERK pathway lead to increases in gene transcription and proliferation, including cytokine gene transcription. The ERK pathway is known to be distinct from the pathway of the present invention; therefore, the discovery that an ERK-independent signal transduction pathway regulates cytokine production is unexpected. The present inventors were the first to appreciate that an MEKK/JNKK-contingent signal transduction pathway, and not an ERK-dependent pathway, regulates cytokine production. Furthermore, the present inventors were the first to appreciate that such an MEKK/JNKK-contingent pathway is activated in mast cells through aggregation of Fcxcex5RI and activation of PI3-kinase (PI3-K). The present inventors were also the first to appreciate the method of regulation of such a signal transduction pathway in order to regulate production of cytokines such as, TNF-xcex1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, G-CSF, GM-CSF, TNF-xcex2, TGF-xcex2, IFN-xcex3, and IFN-xcex1/xcex2, in a hematopoietic cell such as a mast cell, a basophil, an eosinophil, a neutrophil, a T cell, a B cell, a macrophage, a dendritic cell, and a natural killer cell.
One embodiment of the present invention relates to a method to regulate cytokine production by regulating an MEKK/JNKK-contingent signal transduction pathway in a hematopoietic cell. Preferably, such a method comprises regulating one or more of the signal transduction molecule selected from the group of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1 and JNK2.
In one embodiment, an MEKK/JNKK-contingent signal transduction pathway can be regulated by administration of a compound which regulates a signal transduction molecule selected from the group of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1 and JNK2, such that cytokine production is regulated. Preferably, such a compound regulates such a signal transduction molecule by a method such as degrading the molecule, binding an inhibitory compound to the molecule, inhibiting transcription of the molecule, inhibiting translation of the molecule, and inhibiting the interaction of the molecule with another signal transduction molecule.
A preferred embodiment of the present invention relates to a method to regulate cytokine production in a hematopoietic cell expressing Fcxcex5RI by regulating an MEKK/JNKK-contingent signal transduction pathway in such cell. Regulation of an MEKK/JNKK-contingent signal transduction pathway can further comprise regulating other signal transduction pathways that affect the MEKK/JNKK-contingent signal transduction pathway.
Another embodiment of the present invention relates to a method to identify compounds which regulate cytokine production in a hematopoietic cell. Such a method comprises contacting a cell with a putative regulatory compound and determining whether such a compound is capable of regulating cytokine production in a cell by regulating an MEKK/JNKK-contingent signal transduction pathway in the cell.
Yet another embodiment of the present invention relates to a kit for identifying compounds which regulate cytokine production by regulating an MEKK/JNKK-contingent signal transduction pathway.
Another embodiment of the present invention relates to a method to treat a disease involving cytokine production in an animal by regulating an MEKK/JNKK-contingent signal transduction pathway. In one embodiment, such a treatment involves administering to an animal an effective amount of a compound which interacts with a signal transduction molecule in an MEKK/JNKK-contingent signal transduction pathway such that cytokine production is regulated.
A preferred embodiment of the present invention relates to a method to treat allergic inflammation by regulating cytokine production. Such regulation of cytokine production is effected by regulation of an MEKK/JNKK-contingent signal transduction pathway.
Yet another embodiment of the present invention relates to a compound for regulating cytokine production. Such a compound interacts with a signal transduction molecule in an MEKK/JNKK-contingent signal transduction pathway in a manner effective to regulate cytokine production.
Another embodiment of the present invention relates to a cell used in a method to identify compounds capable of regulating cytokine production, comprising a cell having at least one heterologous mammalian nucleic acid sequence encoding at least one protein involved in an MEKK/JNKK-contingent signal transduction pathway. A preferred embodiment relates to a method of using such a cell to screen putative regulatory compounds for their ability to regulate cytokine production in said cell.